1. Field of the Invention
This invention resides in the field of imaging of microarrays by optical excitation of materials in the arrays and detection of the emissions resulting from the excitation, and particularly in systems for conveying excitation light to microarrays.
2. Description of the Prior Art
Numerous devices are commercially available for reading DNA microarrays as well as microarrays of other materials. Examples of these devices are the Chip Scanner technologies of Affymetrix, Inc., Santa Clara, Calif., USA, confocal laser scanners of Agilent Technologies, Palo Alto, Calif., USA, the GenePix microarray scanners of Axon Instruments, Inc., Union City, Calif., USA, the DNAscope(trademark) confocal scanners of GeneFocus, Waterloo, Ontario, Canada, the GeneTAC(trademark) microarray analyzers of Genomic Solutions, Inc., Ann Arbor, Mich., USA, the ScanArray(trademark) Microarray Analysis Systems of Packard BioScience Company, Meriden, Conn., USA, and laser scanning systems of Virtek Vision Corporation, Waterloo, Ontario, Canada.
These devices utilize a lens and mirror system that focuses excitation light from one or more lasers onto individual spots in an array of spots. The emission light from each spot is collected by a lens system with optical bandpass emission filters and one or more photomultiplier tube (PMT) detectors. An image of the array is formed by scanning the narrow excitation source beam and the emission collection optics over the sample. This is achieved by moving either the optics or the sample. This mechanism suffers limitations in sensitivity and in the level of detection and resolution of the resulting image. Systems with a common laser path generally suffer from dichroic optical filter efficiency problems, since rejection of the very strong excitation light is accompanied by rejection of a substantial portion of the much weaker emission light. Further inefficiencies arise from the laser scanning systems in these devices which use confocal pinholes to remove undesirable background, including crosstalk from neighboring array spots, since this reduces the signal light as well.
Devices are also available that deliver broadband white light onto a large area of the sample, and utilize a lens system with optical bandpass emission filters and a CCD detector to collect and quantify the emission light. In one such device, which is available from Applied Precision, Inc. of Issaquah, Wash., USA, and is described in International Patent Publication No. WO 00/62549, international publication date Oct. 19, 2000, a small area (a few square millimeters) is illuminated by a fiber optic ring illuminator. The illuminator is a multiple optical fiber bundle with a working distance ranging from several tenths of an inch to several inches to flood a large number of microarray spots with uniform illumination. Images are formed in panels, each containing all of the microarray spots that were simultaneously illuminated by the fiber bundle. Different image panels representing different portions of the sample are obtained by moving the sample relative to the optical system, and a sufficient number of image panels are collected in this manner to encompass the entire sample. Adjacent panels are then joined or xe2x80x9cstitchedxe2x80x9d together to form a complete image of the sample. Formation of the complete image in this manner requires an expensive precision motion stage, and the stitching is a complicated and laborious process, limiting the quality of the data that this device can generate. A similar device, available from Alpha Innotech Corporation of San Leandro, Calif., USA, floods the entire sample with light and uses a CCD camera to obtain an image of the entire microarray. Current commercially available CCD detectors are of limited resolution, however. In both the Applied Precision and Alpha Innotech devices, crosstalk occurs between array spots.
The present invention resides in illumination and detection systems for microarrays, the systems supplying excitation light through one or more optical fibers, each transmitting excitation light from one or more excitation light sources to a single spot in the array. Emission light generated by each spot is collected, preferably without the use of an optical fiber, by a collimating lens and converted by a detector to a signal that can be processed by conventional imaging software. The collimating lens is preferably used in conjunction with an optical filter. Further signals are obtained by rastering either the microarray or the optical system, and the various signals are compiled by imaging software to form an image of the entire microarray. This system is applicable to any microarrays that utilize labels that emit a signal upon optical excitation. Preferred labels are fluorophores and fluorescent emissions, but the invention extends as well to phosphors and other types of optically excitable labels known to those familiar with biochemical assays.
The optical system is arranged such that the direction of travel of the excitation light differs from the direction along which the emission light is collected, i.e., the two paths do not have a common axis. The optical fiber is configured to illuminate the spot directly, preferably at an acute angle relative to the axis normal to the microarray. The angle of incidence of the excitation light is preferably an acute angle as well. The illumination fiber is configured such that the beam emerging from the fiber is either non-diverging or only minimally diverging.
Preferred light sources are those supplying ultraviolet, visible, or near-infrared light, optically coupled with the optical fiber so that substantially all of the light from the light source enters the fiber for transmission to the spot to be illuminated. The result is substantially no loss of intensity between the light source and the spot. The optical fiber may be a simple fiber or one that includes a collimator, an optical filter, or a collimator-filter-collimator assembly, or any other optical elements or components that process the light in various ways that will enhance its use for particular applications and assays. The output tip of the fiber is preferably shaped to reduce the divergence of the emerging light beam.
Systems in accordance with this invention offer numerous advantages over the prior art. By illuminating only the test area of interest, these systems eliminate crosstalk and maximize the signal of interest. Furthermore, by separating the excitation and emission light paths, systems in accordance with this invention limit the excitation and emission optics to a single function each, thereby permitting individual optimization of these two optical systems. This leads to maximal signal collection and superior performance for any given level of detection and sensitivity. In systems in which the illumination fiber and the emission collection optics are at different angles relative to the normal axis, very little of the excitation light is detected in the emission light path, and a maximal signal-to-noise ratio is achieved. Systems in accordance with this invention thus provide a means of directing excitation light to each individual spot of a microarray in a highly efficient manner while minimizing crosstalk and optimizing the collection of emission light with the highest sensitivity and resolution. Furthermore, excitation systems of this invention require no additional lenses or other optical elements between the excitation fiber and the microarray, and can thus avoid the losses in light intensity that are often caused by these additional elements. In aspects of this invention that are directed to an optical fiber optically coupled directly to an LED or SLD light source, the coupled product is inexpensive, durable, and compact, and delivers bright light while generating minimal heat. This simplified yet highly efficient design presents advantages for packaging, cost and size reduction.